Despite detection of tumor-specific T cells in the peripheral blood and bone marrow of cancer patients, these T cells are unable to mediate total regression of bulky tumors. Immunotherapeutic approaches to expand and activate these T cells rarely result in total tumor regressions or cures. In cancer patients, many factors may contribute to ineffective immunity against tumors including regulatory immune cells such as CD4*CD25* regulatory T cells and GDI 1b* Gr1* immature myeloid cells. In these studies, we intend to decrease immune mediated suppression and delete unresponsive host anti-tumor T cells in tumor bearing mice by treatment with total body irradiation (TBI). We intend to reconstitute the immune system with new T cells by adoptive transfer of naive T cells. We will educate these T cells with peptide-pulsed dendritic cell (DC) vaccination. Initial studies have shown that this strategy leads to a delay in tumor growth but does not enhance survival in melanoma bearing mice. In order to improve this model, the reconstitution and activity of \wo known immune inhibitory populations, CD4*CD25*foxp3* T cells and CD11b*Gr1* immature myeloid cells, will be examined. By depleting these populations, we will determine the effects, if any, these inhibitory cells have on the development of anti-tumor immunity. In addition, strategies that lead to the development of anti-tumor memory T cells and long-term immunity will be explored, including adoptive transfer of tumor reactive T cells. Dendritic cells (DC) are potent antigen presenting cells required for the induction of effective anti-tumor primary and memory T cell responses. I have shown that targeting tumors to cell-surface receptors on DC can lead to enhanced anti-tumor T cell responses and combination therapy with bacterial derived immunoadjuvants can improve DC vaccination against tumors. I hypothesize that regression of established melanoma will require multiple immunotherapeutic approaches for the induction of effective anti-tumor T cell activity. Specifically, I intend to implement therapies for the induction of potent anti-tumor T cell responses against melanoma through the following Specific Aims: 1. To investigate experimentally the mechanisms of immune regulation induced after lymphopenia; 2.To examine the efficacy of DC-based vaccination in the setting of lymphopenia by transfer of T cells enriched in CDS* memory T cells; 3. To determine the efficacy of dendritic cell vaccination and CpG adjuvant therapy in combination with irradiation and adoptive T cell transfer in a murine melanoma model. The experimental studies outlined in this application are designed to continue successful preclinical efforts to generate immunization strategies against melanoma. These finding could have significant translation to human clinical immunotherapeutic trials.